Project Summary The field of structural biology has transformed our understanding of macromolecules, their interactions with other molecules, both small and large, and their functional roles in biology. The ability to determine structures in atomic detail (high resolution) in the past was limited to the use of X-ray crystallography and Nuclear Magnetic Resonance (NMR). More recently, cryo-electron microscopy (cryo-EM) has emerged as a method that is capable of producing high-resolution (near atomic resolution) structures of macromolecules, thereby transforming biology in much the same way that X-ray crystallography and NMR did in the past by providing the ability to determine structures of large macromolecules, or macromolecular complexes that have proven difficult to crystallize. These advances, often called the ?resolution revolution?, have created drastic shifts in the landscape of structural biology where many investigators (mostly X-ray crystallographers) are eager to adopt the new cryo-EM technologies as a primary method of choice or/and compliment to existing approaches. We, investigators at Indiana University School of Medicine (IUSM), are the one of these new wave of users, who are eager to adopt the new cryo-EM technologies. The major user group is supported by 10 R01s, one P01, and two U01 from the NIH. Despite the large potential group of EM users, currently, IUSM does not have any cryo-EM. Moreover, in the entire state of Indiana, there is currently one state-of-the-art high-end cryo-electron microscope, the Titan Krios, dedicated to biological samples, located at Purdue University. This instrument is constantly oversubscribed, which led to a shortage of available time for many users. In order to solve this problem, we formed the Indiana EM consortium with five members from academic institutions including Indiana University School of Medicine (IUSM), Indiana University Bloomington (IUB), and one industrial partner (Eli Lilly Co.). This consortium has secured the funding necessary to purchase a Titan Krios, which will be housed in Purdue. IUSM users will have 31% and IUB will have 6% of the available time. As the Krios will be accessible to IUSM investigators, a lack of cryo-EM in IUSM that can be used for screening cryo- samples for high-resolution data collection on our consortium funded Krios, becomes a paramount problem. To bridge IUSM investigators to the Krios, we are requesting funds for a high-resolution, autoloader based 200KV FEG cryo-electron microscope Glacios, which is uniquely designed for screening cryo-samples. High quality samples identified by Glacios will be used to collect high-resolution datasets on our EM consortium Titan Krios or could be sent to the NIH funded three Service centers as well. The resulting system will provide a maximum throughput cryo-sample screening system, which will serve as a ?Bridge to Krios? by enabling us to collect high-resolution images for three-dimensional reconstruction of macromolecular complexes.